This research project aims to increase our understanding of the mechanisms of action and resistance to methotrexate (MTX), a universal component of therapies for children with acute lymphoblastic leukemia (ALL). The long-term objectives are to incorporate this knowledge in the design of ALL trials based on novel molecular targets and to improve event-free survival (EFS) for patients with resistant phenotypes. Even though the antifolate MTX is widely used in ALL therapies, important unanswered questions remain with respect to molecular determinants of drug resistance, heterogeneity of clinical response, optimal dose(s) and schedule(s). It has been demonstrated that lymphoblast accumulation of MTX polyglutamates (MTX-PGs) correlates with clinical outcome. Metabolism to MTX-PGs depends on serum MTX concentration, transport across the cell membrane, and more important the cell lineage-specific expression of Folyl-gamma-polyglutamate Synthetase (FPGS). Non-random translocations that characterize ALL clones are important predictors of clinical outcome and characterize ALL subtypes that exhibit significant heterogeneity of FPGS expression. Due to their effects on gene transcription and cell cycle control, non-random translocations may alter drug metabolism and resistance. We hypothesize that molecular mechanisms associated with non-random translocations lead to differences in metabolism to MTX-PGs by altering lymphoblast FPGS expression. We propose these translocations represent molecular "pathways" present in leukemic clones that result in the heterogeneity of FPGS expression, patterns of MTX metabolism, and clinical response to MTX seen in childhood ALL subtypes. In addition, single nucleotide polymorphisms (SNPs) of FPGS have been recently identified in an ethnically diverse panel of individuals. We propose these SNPs also contribute to the heterogeneity of PFGS expression in ALL, and their prevalence and physiologic impact will be investigated. In addition, drug-induced mutations leading to in vitro resistance to anti-folates have been described. Consequently, we also hypothesize that drug-induced mutations of FPGS can be detected at the time of relapse from ALL and may represent a novel mechanism of resistance after relapse. More important, because these genetic abnormalities are not present in normal hematopoietic cells, they offer selective targets for gene therapy or other molecular approaches in ALL.